Process for decreasing adiposity using vitamin a as a dietary supplement

ABSTRACT

A process is provided for reducing adiposity in an animal such as a companion animal by feeding the animal an effective amount of Vitamin A for a time sufficient to reduce adiposity in the animal. Preferably, the such effective amount comprises from about 50,000 IU to about 1,000,000 IU of Vitamin A per kilogram of diet. Such an effective amount provides sufficient Vitamin A to decrease accumulation of body fat, increase UCP1 levels, and decrease serum leptin levels in the animal.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application Ser.No. 60/081,969, filed Apr. 16, 1998.

BACKGROUND OF THE INVENTION

This invention relates to a pet food supplement and process fordecreasing adiposity in animals, and more particular, to a pet foodsupplement which includes beneficial amounts of Vitamin A in theanimal's diet.

Obesity is extremely prevalent in many species including humans, dogs,cats and horses. For example, 20 to 40% of dogs and humans have beenestimated to be overweight or obese. Traditionally, high fiber dietshave been used to combat obesity. However, high fiber diets are oftenassociated with several undesirable side effects including decreasedpalatability of food, increased stool volume, increased defecationfrequency, poor skin and hair, improper mineral balance, and decreasedfood digestibility.

An alternative way to control weight is to induce energy expenditure inan individual. Changing the metabolism of fat tissue may regulate energyexpenditure. Fat tissue is generally categorized as white adipose tissue(WAT) or brown adipose tissue (BAT). Energy expenditure, in part, isregulated by BAT. Within BAT is uncoupling protein (UCP)-1. UCP1 is aproton carrier that, upon activation, causes the uncoupling ofrespiration from oxidative phosphorylation, thus causing increasedenergy expenditure from the body through heat generation. It is knownthat all-trans-retinoic acid (RA), one of the active metabolites ofvitamin A, can induce the gene expression of UCP1 in brown adiposetissue (BAT) of rats. Due to its function in energy expenditure andenergy balance, BAT has been implicated to play an important role in thecontrol of obesity.

Another gene that has been recently identified to play an important rolein energy homeostasis is the ob gene. The product of the ob gene,leptin, is primarily produced in white adipose tissue (WAT). Leptin isbelieved to be the signal for the level of adiposity, and this hormoneboth suppresses food intake and increases energy expenditure. However,there have been few studies on diet compositions designed to induceenergy expenditure as a means to control obesity.

Accordingly, there is still a need in the art for decreasing adiposityin animals including companion animals such as dogs, cats, and horses.

SUMMARY OF THE INVENTION

The present invention addresses the need for reducing adiposity byproviding a process for feeding an animal an effective amount of VitaminA for a time sufficient to reduce adiposity in the animal. By “reduceadiposity,” we mean that for a given animal ingesting a given amount offood, the percentage of body fat in the animal will be lower when theanimal is provided with the effective amount of Vitamin A as comparedwith an animal ingesting the same amount of food, but without Vitamin Asupplementation. The Vitamin A may be provided to the animal either as asupplement or contained in a diet fed to the animal. Such a supplementmay be in the form of a pill or capsule, a treat or biscuit, or anyother edible form. By “diet” we mean the food or drink regularlyconsumed by the animal.

When supplied as a supplement, the supplement preferably comprises fromabout 50,000 IU to about 1,000,000 IU of Vitamin A per kilogram of diet,more preferably, from about 50,000 IU to about 500,000 IU of Vitamin Aper kilogram of diet, and most preferably, from about 50,000 IU to about150,000 IU of Vitamin A per kilogram of diet. These amounts are over andabove the amount of Vitamin A which may be present in other componentsof the diet of the animal.

The supplement is preferably fed to the animal in an amount of about50,000 IU to about 1,000,000 IU of Vitamin A per day (based on a diet of1,000 g per day), or about 5,000 IU to about 100,000 IU of Vitamin A perday (based on a diet of 100 g per day). The Vitamin A may be provided inthe supplement as retinol, and provides sufficient Vitamin A to resultin a reduction in adiposity of the animal.

The supplement may be fed to companion animals including dogs, cats, andhorses. In addition to reducing adiposity in animals, the supplement mayalso be used to increase UCP1 gene expression, suppress leptin geneexpression, and suppress serum leptin levels. The supplement may also beused to help prevent obesity, promote weight loss, and may also be usedto minimize age-related increases in body fat and diabetes-associatedincreases in body fat.

Accordingly, it is a feature of the invention to provide a pet foodsupplement or diet and process for reducing adiposity by providing aneffective amount of Vitamin A in the diet of the animal. This, and otherfeatures and advantages of the present invention, will become apparentfrom the following detailed description, accompanying drawings, andappended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of serum leptin levels (ng/ml) in rats fed a normaldiet and a vitamin A supplemented diet for 8 weeks;

FIG. 2 is a graph of leptin mRNA levels (arbitrary units/μg RNA) in ratsfed a normal diet or a Vitamin A supplemented diet;

FIG. 3 is a graph of oxygen consumption (Δml/min/kg^(0.67)) versus timefor rats fed a normal diet or a Vitamin A supplemented diet; and

FIG. 4 is a graph of UCP1 mRNA levels (arbitrary units/μg RNA) for ratsfed a normal diet or a Vitamin A supplemented diet; and

FIG. 5 is a graph showing weight gain in normal weight dogs fed VitaminA.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention uses a supplement or diet which contains a sourceof Vitamin A in an amount of between about 50,000 IU to about 1,000,000IU of Vitamin A per kilogram of diet.

Feeding animals effective amounts of Vitamin A results in a reduction inadiposity (as compared with an animal fed the same diet but withoutVitamin A supplementation), an increase in β3-adrenergic stimulatedoxygen consumption, an increase in UCP1 gene expression in BAT, andsuppression of leptin gene expression and serum leptin levels. Thereduction in adiposity is surprising because the suppression of leptingene expression would be expected to promote obesity by reducing leptinlevels in the animal.

The Vitamin A may be included in a diet which can comprise any suitablepet food formulation which also provides adequate nutrition for theanimal. For example, a typical canine diet for use in the presentinvention may contain about 18-40% crude protein, about 4-30% fat, andabout 4-20% total dietary fiber. However, no specific ratios orpercentages of these or other nutrients are required. The Vitamin A,preferably in the form of retinol, may be blended with such pet foodformulation such that the diet includes from about 50,000 IU to about1,000,000 IU of Vitamin A per kilogram of diet.

In order that the invention may be more readily understood, reference ismade to the following examples which are intended to illustrate theinvention, but not limit the scope thereof.

EXAMPLE 1

Male F-344×BN rats of 5 months of age were obtained from HarlanSprague-Dawley (Indianapolis, Ind.). Upon arrival, rats were examinedand remained in quarantine for one week. Animals were cared for inaccordance with the principles of the Guide to the Care and Use ofExperimental Animals. Rats were housed individually with a 12:12 hlight-dark cycle (07:00 to 19:00 hr). Ambient temperature was 26° C.,thermoneutrality for these rats.

Rats were fed a diet containing either, 8,643 IU retinol/kg diet (normaldiet) or 430,431 IU retinol/kg diet (vitamin A supplemented diet) for aperiod of 8 weeks. Food and water were provided ad libitum. Body weightsand food intake were recorded weekly. Basal whole body oxygenconsumption was measured before starting the diet and then after week 3,6 and 8 on the diet. At the end of 8 weeks, half the rats from eachgroup were challenged with a single dose of the β3 adrenergic specificagonist, CGP-12177 (prepared in pyrogen-free saline, available fromCiba-Geigy, Summit, N.J.) (0.75 mg/kg, i.p.) or saline. The stimulatedwhole body oxygen consumption was measured and the animals sacrificed 4hours after injection.

Oxygen Consumption

O₂ consumption was assessed on up to four rats simultaneously with anOxyscan analyzer (OXS-4; Amniotic Electronics, Columbus, Ohio). Allexperiments were performed on conscious unanesthetized rats during thelight phase of the light-dark cycle. Flow rates were 2 L/min with a 30second sampling time at 5 minute intervals. The temperature wasmaintained at 26° C. Results were expressed on a mass-dependent basis(ml/min/g^(0.67)). The cumulative increase in the CGP12177-stimulatedoxygen consumption was calculated as the average increase over baselinefrom 30 min to 110 min, post injection.

Tissue Harvesting

Rats were sacrificed by cervical dislocation under 90 mg/kgpentobarbital anesthetic. Blood was collected in Vacutainer SST tubes(Becton Dickinson, Franklin Lakes, N.J.) via cardiac puncture using an18-gauge needle followed by perfusion with 60 ml of 0.9% saline.Interscapular brown adipose tissue (IBAT), perirenal white adiposetissue (PWAT), retroperitoneal white adipose tissue (RTWAT), epididymalwhite adipose tissue (EWAT) and liver were excised, weighed, and rapidlystored in liquid nitrogen. The tissues were stored at −70° C. untilanalysis.

Determination of Adiposity Levels

Adiposity was determined by the adiposity index (the sum of the weightsof perirenal WAT, retroperitoneal WAT, and epididymal WAT divided bybody weight×100). This adiposity measure is highly correlated with thepercentage of body fat.

Leptin Radioimmunoassay

Serum leptin levels were measured with a rat leptin radioimmunoassay kit(Linco Research, St. Charles, Mo.).

Serum and Liver Retinol

Total retinol from serum (400 μL) and liver (500 μg) were extracted byaddition of 1 ml hexane with 0.01% BHT in the cold. The hexaneextraction was repeated twice and the extracts pooled and evaporated todryness under nitrogen. Extracted lipids were resuspended in hexanemethanol (1:9) solution containing 0.01% BHT and analysis for retinol byHPLC (Furr et al. 1984).

UCP1 and Leptin mRNA Levels

Total cellular RNA was extracted using a modification of the methoddescribed in Chomczynski and Sacchi, “Single step method of RNAisolation by acid guanidinium thiocyanate-phenolchloroform extraction”,Analytical Biochemistry 162:156-159 (1987). The integrity of theisolated RNA was verified using 1% agarose gels stained with ethidiumbromide. The RNA was quantified by spectrophotometric absorption at 260nm using multiple dilutions of each sample.

The probe to detect leptin mRNA was a 33-mer antisense oligonucleotide(5′GGTCTGAGGCAGGGAGCAGCTCTTGGAGAAGGC), end-labeled using terminaldeoxynucleotidyl transferase. The oligonucleotide was based on a regionof the mRNA downstream from the site of the primary mutation in ob/obmice and synthesized at the University of Florida core facility andverified by Northern analysis as described in Li et al,“Beta(3)-adrenergic-mediated suppression of leptin gene expression inrats,” American Journal of Physiology 272 (1997). The full length cDNAclone for uncoupling protein-1 (UCP1) was provided by Dr. Leslie Kozak,Jackson Laboratory, Bar Harbor, Me. and verified by Northern analysis,as described in Scarpace et al., “Thermoregulation with age: Role ofthermogenesis and uncoupling protein expression in brown adiposetissue”, Proceedings of the Society for Experimental Biology andMedicine 205:154-161(1994). The UCP1 cDNA probe and the full lengthhuman β-actin cDNA probe (Clontech, Palo Alto, Calif.) and the mouse LPLcDNA clone (ATCC. Rockville. Md.) were random prime labeled usingPrime-a-Gene (Promega).

For dot-blot analysis, multiple concentrations of RNA were immobilizedon nylon membranes using a dot-blot apparatus (Biorad, Richmond,Calif.). The membranes were baked at 80° C. for 2 hrs. The bakedmembranes were prehybridized using 25 mM potassium phosphate, 750 mMNaCl, 75 mM Na citrate, 5×Denhardt's solution, 50 μg/ml denatured salmonsperm DNA, and 50% formamide. After incubation for 14-16 hrs. at 42° C.,the membranes were hybridized with ³²P labeled probes in theprehybridization buffer plus 10% dextran sulfate. After hybridizationfor 14-16 hr at 42° C., the membranes were washed and exposed to aphosphor imaging screen for 48 hrs. The latent image was scanned using aPhosphor Imager (Molecular Dynamic, Sunnyvale, Calif.) and analyzed byImage Quant Software (Molecular Dynamics). Intensities per μg totalcellular RNA were calculated by comparison to internal laboratorystandards of WAT or BAT total RNA present on each nylon membrane.

Data Analysis

One way or two way analysis of variance was applied where appropriate.When main effect was significant, Scheffe's post hoc comparison wasapplied.

RESULTS

Weight Gain Food Intake, Body Fat and Vitamin A Status

At the end of the 8 week period on the normal or vitamin A supplementaldiet, the vitamin A status of the animals was evaluated by determiningthe concentration of retinol in serum and liver. Whereas, the serumretinol concentrations were unaffected by vitamin A supplementation, theliver retinol concentration was 10.8 fold greater in the vitamin Asupplemented group, compared with controls (Table 1). This relativedifference in liver retinol was nearly equal to the 13.75 folddifference in the vitamin A contents of the normal and supplementeddiets. The vitamin A supplemented rats, however, did not show any signsof vitamin A toxicity, including depressed growth, occasional bleedingfrom the nose, or partial paralysis of the legs.

TABLE 1 Serum and liver retinol levels in control and dietary Vitamin Asupplemented rats Retinol Diet Serum (ng/ml) Liver (ng/ml) Normal 34 ± 1 498 ± 28 Vitamin A Supplemented 32 ± 1 5406 ± 163* Data represent mean± SE of 15 (normal diet) or 16 (Vitamin A supplemented diet) rats. *p =0.0001 for difference from rats fed the normal diet by one-way ANOVA.

Pre-diet and post-diet body weights as well as the gain in body weightin the control and in the vitamin A supplemented groups was similar(Table 2). Food intake was also unchanged by dietary vitamin Asupplementation (Table 2).

TABLE 2 Food intake and body weight parameters and adiposity in controland dietary Vitamin A supplemented rats Diet Normal Vitamin Asupplemented Daily food intake (g/day) 18.1 ± 0.3 18.1 ± 0.4 Pre dietbody weight (g)  328 ± 6  326 ± 5 Post diet body weight (g)  390 ± 8 386 ± 7 Body weight gain (g) 61.8 ± 5.1 59.3 ± 5.2 BAT weight (mg)  411± 12  430 ± 20 PWAT weight (mg) 1.49 ± 0.06 1.23 ± 0.07* RTWAT weight(mg) 3.80 ± 0.10 3.53 ± 0.13 EWAT weight (mg) 2.98 ± 0.10 2.62 ± 0.09*Adiposity index 2.09 ± 0.04 1.91 ± 0.04* Data represent mean ± SE of 15(control) or 16 (Vitamin A supplemented) rats. *p = 0.005 (PWAT), p =0.013 (EWAT) or p = 0.004 (Adiposity index) for difference from rats fedthe normal diet by one-way ANOVA.

In contrast, there was a significant decrease in PWAT and EWAT weight inthe rats fed the vitamin A supplemented compared with the normal diet(Table 2). In rats on the vitamin A supplemented diet, there was a 17%reduction in the weight of PWAT, and 12% reduction in the weight ofEWAT. The weight of the RTWAT, a third WAT depot was unchanged (Table2). Overall, there was a significant decrease in the adiposity index[(sum of three WAT depots divided by the body weight)×100], in the ratsfed the vitamin A supplemented diet (Table 2). In contrast, there wereno changes in BAT weights, upon vitamin A supplementation (Table 2).

Serum Leptin Levels and Leptin mRNA Levels in PWAT

In general, serum leptin levels reflect body fat content, thus thedecrease in adiposity in the dietary vitamin A supplemented rats shouldresult in a corresponding decrease in serum leptin. Surprisingly, thedecrease in serum leptin in the vitamin A supplemented rats was 65%,much greater than predicted from the 9% decrease in the adiposity index(FIG. 1). This dramatic decrease in serum leptin suggests leptinsynthesis was inhibited.

To assess if there was a suppression of leptin mRNA levels in PWAT uponvitamin A supplementation, leptin mRNA levels were compared in thecontrol animals on the vitamin A supplemented and control diet. Therewas a 44% suppression of leptin mRNA levels per unit of total RNA inrats on the vitamin A supplemented, compared with the normal diet (FIG.2). β3-adrenergic agonists also inhibit leptin gene expression. Tocompare the suppression of leptin gene expression by the β3-adrenergicagonist, CGP-12177 with dietary vitamin A supplementation, at the end ofthe 8 week dietary regimen, each dietary group was administeredCGP-12177 and leptin mRNA levels determined in PWAT 4 hr later. Asexpected, the β3-adrenergic agonist suppressed leptin mRNA levels by 27%in the rats on the normal diet (FIG. 2). In contrast, in the vitamin Asupplemented group, administration of CGP-12177 had no further effect onleptin mRNA levels, compared with the saline treated controls on thesame diet (FIG. 2). There were no changes in the levels of β actin mRNAin BAT in the saline or CGP-12177 treated animals in the two dietarygroups (Table 3).

TABLE 3 UCP2, β₃-adrenergic receptor and β-actin transcript levels withand without CGP-12177 administration in rats fed a normal or Vitamin Asupplemented diet MRNA levels (arbitrary units) Normal Diet Vitamin Asupplemented diet Transcript Saline CGP-12177 Saline CGP-12177 PWAT:UCP2 100 ± 11  109 ± 10  110 ± 14  110 ± 10 β₃AR 100 ± 9.2  105 ± 6.084.0 ± 13.1  112 ± 11 BAT: UCP2 100 ± 13 93.4 ± 12.2  103 ± 5.2   94 ±8.9 β₃AR 100 ± 4 50.9 ± 3.3*  104 ± 15.6 59.8 ± 7.9* β-actin 100 ± 1098.6 ± 7.4 86.1 ± 7.2 93.8 ± 8.0 Data represent mean ± SE of 6-7 ratsper group. β₃AR is β₃-adrenergic receptor. *p = 0.0001 for differencebetween CGP-12177 administration and control rats by two-way ANOVA. p =0.0001 (normal diet) and p = 0.003 (vitamin A supplemented diet) fordifference between CGP-12177 administration and corresponding controlrats.

Oxygen Consumption

The decrease in adiposity with dietary vitamin A supplementation withouta change in food intake suggests an increase in energy expenditure. Toassess if there was enhanced energy expenditure upon vitamin Asupplementation, basal whole body oxygen consumption was measured beforestarting the diet and at weeks 3, 6 and 8 on the diet. There was nochange in basal oxygen consumption between the two dietary groups(normal and vitamin A supplemented) at either week 3, 6, or 8 afterstarting the diet (Table 4).

TABLE 4 Basal whole body oxygen consumption over 8 weeks in rats fed thenormal and Vitamin A supplemental diets Oxygen consumption(ml/min/kg^(0.67)) Normal diet Vitamin A supplemented Pre-diet 12.7 ±0.6 12.8 ± 0.9 Week 3 11.0 ± 0.1 10.8 ± 0.1 Week 6 10.6 ± 0.2 10.4 ± 0.1Week 8 10.4 ± 0.2 10.2 ± 0.2 Data represent mean ± SE of 16 rats in eachgroup. Oxygen consumption was assessed over a one hour period.

To determine if there was an upregulation of the capacity forthermogenesis upon dietary vitamin A supplementation, the β3-adrenergicstimulated increase in oxygen consumption was measured in the twodietary groups at the end of the 8th week. Half the animals in the twodietary groups were administered either saline or the β3-adrenergicspecific agonist, CGP-12177 (0.75 mg/kg, i.p.), and the increase inoxygen consumption measured. Upon administration of CGP-12177, there wasa rapid and sustained increase in oxygen consumption in the rats on thenormal and vitamin A supplemented diets, compared with the correspondingsaline injected rats of the same dietary group. The averaged peakincrease in the CGP-12177-stimulated oxygen consumption (calculated asthe average increase over saline injection from 30 min to 110 min,postinjection) in the rats fed the vitamin A supplemented diet was 14%higher than in the rats fed the normal diet (FIG. 3).

UCP1 mRNA Levels in BAT

To determine if the increase in β3-adrenergic stimulated oxygenconsumption was associated with elevated UCP1 gene expression, theeffects of vitamin A supplementation on UCP1 mRNA levels in BAT weredetermined by comparison of UCP1 mRNA levels in rats on the vitamin Asupplemented and normal diets. There was a 31% increase in UCP 1 mRNAlevels upon dietary vitamin A supplementation (FIG. 4). To assess ifthere was an increased capacity to respond to β3-adrenergic agoniststimulation in the dietary vitamin A supplemented rats, changes in UCP1mRNA levels were compared in the CGP-12177 administered animals on thevitamin A supplemented and normal diets. Upon administration ofCGP-12177, there was a significant but similar increase in UCP1 mRNAlevels in rats on both the vitamin A supplemented and normal diets (FIG.4). There were no changes in the levels of β actin mRNA in BAT in thesaline or CGP-12177 treated animals in the two dietary groups (Table 4,above).

UCP2 mRNA Levels in BAT and PWAT

To determine if increases in the gene expression of UCP2 could have alsocontributed to the increase in CGP- 12177-stimulated oxygen consumptionin vitamin A supplemented animals, UCP2 mRNA levels were measured inPWAT and BAT. There were no changes in the levels of UCP2 mRNA in PWATbetween the two dietary groups (Table 4, above). Furthermore, treatmentwith CGP 12177 had no effect on the levels of UCP2 mRNA in PWAT ineither of the two dietary groups of animals. Similar to PWAT, there wereno changes in UCP2 mRNA levels in BAT, in any of the four experimentalgroups (Table 4).

β₃-adrenergic Receptor mRNA Levels in PWAT and BAT

To determine if the increase in β3-adrenergic stimulated oxygenconsumption or suppression of leptin mRNA level was associated with anincrease in β3-adrenergic receptor gene expression, the effects ofvitamin A supplementation on β3-adrenergic receptor mRNA levels weremeasured in BAT and PWAT. There were no changes in the levels ofβ3-adrenergic receptor mRNA in either the saline or CGP-1 2177 treatedanimals on either the normal or vitamin A supplemented diet (Table 4).Similarly, in BAT, there were no changes in the levels of β3 adrenergicreceptor mRNA in the saline treated animals on the vitamin Asupplemented diet compared with the normal diet (Table 4). Upontreatment with CGP-12177 for 4 h, there was a similar (50%) downregulation of 133-adrenergic receptor mRNA levels in animals on thevitamin A supplemented and normal diets, compared with the correspondingsaline treated animals from the two dietary groups (Table 4).

The results demonstrate that dietary vitamin A supplementation decreasesadiposity in rats. This decrease in adiposity was associated with anincrease in UCP1 gene expression but a decrease in a leptin geneexpression. The decrease in adiposity was not associated with any changein food consumption suggesting it was a result of a redistribution ofbody composition or an increase in energy expenditure. Basal levels ofwhole body oxygen consumption were unchanged with dietary vitamin Asupplementation, but β3-adrenergic stimulated oxygen consumption wasgreater in the rats supplemented with vitamin A compared with those on anormal diet. This suggests that the capacity for nonshiveringthermogenesis in BAT was increased in the vitamin A supplemented rats.

Thermogenesis in BAT is mediated by norepinephrine activation ofsympathetically innervated β3-adrenergic receptors. The β3-adrenergicsignal transduction pathway serves both to activate BAT mitochondrialUCP1 and induce new synthesis of this protein. Activated UCP1 uncouplesmitochondria, allowing high rates of substrate oxidation and heatproduction without phosphorylation of adenosine 5′ diphosphate. Thedemonstration of an increase in basal UCP1 gene expression followingdietary vitamin A supplementation suggests an upregulation in thecapacity for thermogenesis in BAT. Furthermore, the increase inβ3-adrenergic stimulated oxygen consumption suggests that an increase inenergy expenditure may be contributing to the decrease in adiposityfollowing dietary vitamin A supplementation.

In addition to β3-adrenergic agonists, leptin is another agent thatincreases thermogenesis in BAT. The most dramatic changes observedfollowing dietary vitamin A supplementation were decreases in both serumleptin and leptin gene expression in PWAT. These changes were notconsistent with either the decrease in adiposity or the increase in UCP1gene expression. Leptin administration decreases food intake andincreases energy expenditure. Despite the fall in serum leptin, foodintake was unchanged and energy expenditure was increased. While notwishing to be bound by any particular theory, the leptin-inducedincrease in energy expenditure is believed to be a result of an increasein sympathetic nerve activity to BAT resulting in an increase in UCP1gene expression. However, despite the fall in serum leptin, UCP1 geneexpression increased.

Along with UCP1, the effect of vitamin A supplementation was examined onanother transcript that may be involved in energy balance, UCP2. Thisuncoupling protein has 59% homology with UCP1 and 73% homology withUCP3. Similar to UCP1, UCP2 can partially uncouple mitochondrialrespiration. The expression of UCP2, unlike UCP1, is not limited to BAT,and this protein is widely expressed in many tissues, including WAT,heart, and muscle in both rodents and humans. This experiment found nochanges in UCP2 gene expression in PWAT with dietary vitamin Asupplementation.

The salient findings of decreased adiposity, increased UCP1 geneexpression, and decreased leptin gene expression are similar to what isobserved following administration of a β3-adrenergic agonist.β3-adrenergic agonists suppress leptin gene expression in white adiposetissue, greatly enhance UCP1 gene expression in BAT, and decreaseadiposity in rats. Thus, one possibility is that the effects of vitaminA supplementation may be mediated by enhanced β3-adrenergicresponsiveness. No conclusive evidence of enhanced β3-adrenergicreactivity in the vitamin A supplemented rats was found, however. InBAT, β3-adrenergic receptor gene expression was unchanged with vitamin Asupplementation. UCP1 gene expression was unchanged with vitamin Asupplementation. UCP1 gene expression was elevated in vitamin Asupplemented rats but this was most likely a direct result of retinoicacid, an active metabolite of vitamin A. It has been previouslydemonstrated that retinoic acid administration increases UCP1 geneexpression. This induction is mediated by a retinoic acid-responsiveelement in promoter region of the UCP1 gene. Vitamin A and β3-adrenergicagonist induced increases in UCP1 gene expression following CGP-12177stimulation was the same in rats on both the normal and vitamin Asupplemented diets. In white adipose tissue, there was also no change inβ-adrenergic receptor mRNA levels with vitamin A supplementation. Incontrast, the suppression of leptin gene expression by the vitamin Asupplemented diet could be the result of enhanced β3-adrenergicreactivity. Suppression of leptin gene expression by the vitamin Asupplementation and by β3-adrenergic agonist administration were notadditive. Moreover, in the vitamin A supplemented rat, there was nofurther decrease in leptin gene expression with CGP-12177administration. This suggests that the two treatments share a commonmechanism or that with vitamin A supplementation, the leptin geneexpression is suppressed to maximum extent possible and that furthersuppression by β3-adrenergic stimulation is ineffective.

It also was found that basal whole body oxygen consumption was unchangedby the vitamin A supplemented diet, however, β3-adrenergic stimulatedoxygen consumption was increased. The latter could represent enhancedβ3-adrenergic function or could reflect the vitamin A-induced increasein UCP1 gene expression. Collectively, these data suggest that theeffects of vitamin A on adiposity, UCP1 and leptin gene expression arenot due to increases in endogenous stimulation of β3-adrenergicreceptors, but the increased oxygen consumption in response to CGP-12177and the failure of this β3-adrenergic agonist to suppress leptin geneexpression in the vitamin A supplemented rat may involve alteredβ3-adrenergic signal transduction in BAT and PWAT.

The decrease in adiposity with vitamin A supplementation was highlysignificant (9%). This amount of decrease was surprising consideringthese were young lean F-344×BN rats that are not prone to obesity. Moresurprising, is that this decrease in adiposity was despite the greaterthan 65% decrease in serum leptin. Moreover food intake was unchanged.This suggests that leptin may not be the primary regulator of foodintake or energy expenditure under these conditions. Furthermore,vitamin A supplementation may provide a convenient method to reduceserum leptin under conditions where it is abnormally elevated. Bothobesity and the increase in body weight with aging are associated withelevated levels of serum leptin and leptin resistance. Moreover, theincreased levels of leptin with obesity may be contributing to thediabetes caused by obesity. Recent studies have indicated that leptinmay impair insulin action. Leptin inhibits the basal and glucosemediated insulin secretion from isolated pancreatic islets of bothnormal rats and ob/ob mice. Leptin also impairs the metabolic action ofinsulin in isolated rat adipocytes including glucose transport andlipogenesis. It has been indicated that leptin inhibits insulin signaltransduction in human hepatic cells. However, this inhibition could notbe duplicated in a hepatic cell line transfected with the leptinreceptor. Thus, the elevated leptin levels in obese individuals may beharmful. Vitamin A supplementation may be one method to restore bothleptin and insulin responsiveness under conditions of obesity andelevated serum leptin.

In summary, the data show that dietary vitamin A supplementationdecreases adiposity, increases β3-adrenergic stimulated oxygenconsumption and UCP1 gene expression in BAT, and suppresses leptin geneexpression and serum leptin levels. Thus, dietary vitamin Asupplementation has a role as an anti-obesity treatment or as one methodto lower abnormal elevated serum leptin levels in animals.

EXAMPLE 2

To evaluate the influence of high amounts of vitamin A in the diet ofdogs, an experiment was designed utilizing normal weight (12.1 kg)beagles. Thirty dogs were fed a diet minimally sufficient in vitamin A(9 KIU/kg) for two months. The dogs were then divided into two groups.Each group was fed one of two diets that contained a high amount of fat(24.4% DMB). The only difference in the diets was the amount of vitaminA (control=9 KIU/kg vs. high vitamin A=129 KIU/kg). The animals wereallowed to consume these diets free choice for a 13-week period.Animals' body weights were assessed weekly and food intakes wereassessed daily. Body composition was assessed using dual energy x-rayabsorptiometry just before, during, and after consuming the high fatdiets. Animals fed the high vitamin A diet gained less weight than thecontrol fed animals (FIG. 5). High vitamin A consumption helped decreasebody fat while maintaining muscle mass (Table 5). As can be seen fromthe table, body fat tends to decline and lean body mass increases asanimals are fed increased amount of vitamin A. These results demonstratethe value of feeding increased vitamin A to help remove body fat (i.e.,reduce adiposity) and prevent obesity.

TABLE 5 Body Composition of Dogs Fed Control or Supplemental Vitamin ATime Body Lean Body Bone Mineral Diet Measured Fat (%) Mass (%) Content(%) Control Baseline 30.9 67.3 1.8 High Vitamin A Baseline 31.4 66.8 1.8Control Mid-point 41.7 56.7 1.6 High Vitamin A Mid-point 40.8 57.6 1.7Control End 44.3 54.2 1.6 High Vitamin A End 43.1 55.3 1.6

While certain representative embodiments and details have been shown forpurposes of illustrating the invention, it will be apparent to thoseskilled in the art that various changes in the methods and apparatusdisclosed herein may be made without departing from the scope of theinvention, which is defined in the appended claims.

What is claimed is:
 1. A process for reducing adiposity of a companionanimal comprising the step of feeding said companion animal an effectiveamount of Vitamin A for a time sufficient to reduce adiposity in saidcompanion animal.
 2. A process as claimed in claim 1 in which saidVitamin A is fed to said companion animal as a supplement in an amountof about 50,000 IU to about 1,000,000 IU of Vitamin A per day.
 3. Aprocess as claimed in claim 1 in which said Vitamin A is fed to saidcompanion animal as a supplement in an amount of about 5,000 IU to about100,000 IU of Vitamin A per day.
 4. A process as claimed in claim 1wherein said companion animal is a horse.
 5. A process as claimed inclaim 1 wherein said companion animal is a dog.
 6. A process as claimedin claim 1 wherein said companion animal is a cat.
 7. A process forreducing adiposity of a companion animal comprising the step of feedingsaid companion animal a diet containing an effective amount of Vitamin Afor a time sufficient to reduce adiposity in said companion animal.
 8. Aprocess as claimed in claim 7 in which said diet includes from about50,000 IU to about 1,000,000 IU of Vitamin A per kilogram of diet.
 9. Aprocess as claimed in claim 7 in which said diet comprises about 18 to40% crude protein, about 4 to 30% fat, and about 4 to 20% total dietaryfiber.